Engineered microorganisms having resistance to ionic liquids

ABSTRACT

The present invention provides for a method of genetically modifying microorganisms to enhance resistance to ionic liquids using a yeast Major Facilitator Superfamily (MFS), or a  Salmonella  MFS SmvA Pump or SmvR Regulator, a Small Multidrug Resistance Family (SMR), or  Saccharomyces cerevisiae  YDR090C, polypeptide, host cells genetically modified in accordance with the methods, and methods of using the host cells in a reaction comprising biomass that has been pretreated with ionic liquids.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application Ser. No. 62/001,014, filed on May 20, 2014, which is hereby incorporated by reference.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by the United States Department of Energy and Award No. EEC-0540879 awarded by the National Science Foundation. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to microbial resistance to ionic liquids.

BACKGROUND OF THE INVENTION

Biomass feedstock pretreatment with ionic liquids (ILs) reduces recalcitrance of lignocellulose to degradation. However, ILs are often toxic to microorganisms used subsequently for saccharification and fermentation and the low levels of IL that can remain after pretreatment inhibit the growth of organism used in saccharification and/or fermentation reactions. There is therefore a need to improve IL tolerance of such organisms. This invention addresses this need.

SUMMARY OF THE INVENTION

In one aspect, this invention relates to a method of increasing tolerance of a microorganism, e.g., Escherichia coli, to ILs. The method comprises engineering the microorganism to express a gene encoding a yeast Major Facilitator Superfamily (MFS) protein, or a Salmonella MFS SmvA Pump or SmvR Regulator. The invention further provides recombinant microorganisms engineered to express a yeast MFS protein, or Salmonella MFS SmvA Pump or SmvR Regulator, and methods employing such microorganisms to process biomass.

In one aspect, the invention provides a recombinant microorganism having resistance to ionic liquids, e.g., an ionic liquid where the anion is Cl⁻ or acetate, wherein the microorganism comprises a heterologous gene encoding a yeast MFS, or Salmonella MFS SmvA Pump or SmvR Regulator, polypeptide wherein the yeast MFS polypeptide has at least 70% identity, or at least 75%, 80%, or 85% identity, to SEQ ID NO:21, or SEQ ID NOs:1 or 2. In some embodiments, the yeast MFS, or Salmonella MFS SmvA Pump or SmvR Regulator, polypeptide has at least 90% identity, or at least 95% identity, or at least 99% identity, to SEQ ID NO:21, or SEQ ID NOs: 1 or 2. In some embodiments, the polypeptide comprises SEQ ID NO:21, or SEQ ID NOs:1 or 2. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the microorganism is also genetically modified to express a tet repressor protein that binds to the tet repressor sequence. In some embodiments, the microorganism is a yeast or filamentous fungi. In some embodiments, the heterologous gene encoding the yeast MFS, or Salmonella MFS SmvA Pump or SmvR Regulator, polypeptide is integrated into the genome of the microorganism. In some embodiments, the heterologous gene encoding the yeast MFS, or Salmonella MFS SmvA Pump or SmvR Regulator, polypeptide is present on an extrachromosomal autonomously replicating sequence present in the microorganism.

In a further aspect, the invention provides a method of modifying a microorganism to have resistance to ionic liquids, e.g., ionic liquids where the anion is Cl⁻ or acetate, the method comprising introducing an expression cassette into the microorganism, wherein the expression cassette comprises a gene encoding a yeast MFS, or Salmonella MFS SmvA Pump or SmvR Regulator, polypeptide that has at least 70% identity, or at least 75%, 80%, or 85% identity, to SEQ ID NO:21, or SEQ ID NOs:1 or 2. In some embodiments, the yeast MFS, or Salmonella MFS SmvA Pump or SmvR Regulator, polypeptide has at least 90% identity, or at least 95% identity, or at least 99% identity, to SEQ ID NO:21, or SEQ ID NOs:1 or 2. In other embodiments the polypeptide comprises SEQ ID NO:21, or SEQ ID NOs:1 or 2. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the method further comprises engineering the microorganism to express a tet repressor that binds to the tet repressor sequence. In some embodiments, the method comprises engineering bacteria, such as Escherichia coli. In other embodiments, the method comprises engineering a yeast or filamentous fungi. In some embodiments, the method comprises integrating the heterologous gene encoding the yeast MFS, or Salmonella MFS SmvA Pump or SmvR Regulator, polypeptide into the genome of the microorganism.

In a further aspect, the invention provides a composition comprising a microorganism of the invention comprising a heterologous yeast MFS, or Salmonella MFS SmvA Pump or SmvR Regulator, gene that is capable of expressing yeast MFS, or bacterial MFS. In some embodiments, the composition is a reaction mixture that comprises an ionic liquid and the microorganism of the invention. In some embodiments, the reaction mixture further comprises biomass.

In an additional aspect, the invention provides a method of increasing the yield of soluble sugar from a biomass, the method comprising incubating biomass pretreated with a microorganism genetically modified to comprise a heterologous yeast MFS, or Salmonella MFS SmvA Pump or SmvR Regulator, gene in an enzymatic hydrolysis reaction. The invention additionally provides a method of increasing the yield from a reaction in which soluble sugars are a source of carbon, e.g., a fermentation reaction that produces a biofuel, such as an alcohol, the method comprising incubating biomass pretreated with ionic liquid with a microorganism of the invention in a reaction.

In one aspect, this invention relates to a method of increasing tolerance of a microorganism, e.g., Escherichia coli, to ILs. The method comprises engineering the microorganism to express a gene encoding a Small Multidrug Resistance Family (SMR) protein. The invention further provides recombinant microorganisms engineered to express a SMR protein and methods employing such microorganisms to process biomass.

In one aspect, the invention provides a recombinant microorganism having resistance to ionic liquids, e.g., an ionic liquid where the anion is Cl⁻ or acetate, wherein the microorganism comprises a heterologous gene encoding a SMR polypeptide, wherein the SMR polypeptide has at least 70% identity, or at least 75%, 80%, or 85% identity, to one amino acid sequence of the group consisting of SEQ ID NOs:3-20. In some embodiments, the SMR polypeptide has at least 90% identity, or at least 95% identity, or at least 99% identity, to one amino acid sequence of the group consisting of SEQ ID NOs: 3-20. In some embodiments, the polypeptide comprises one amino acid sequence of the group consisting of SEQ ID NOs: 3-20. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the microorganism is also genetically modified to express a tet repressor protein that binds to the tet repressor sequence. In some embodiments, the microorganism is a yeast or filamentous fungi. In some embodiments, the heterologous gene encoding the SMR polypeptide is integrated into the genome of the microorganism. In some embodiments, the heterologous gene encoding the SMR polypeptide is present on an extrachromosomal autonomously replicating sequence present in the microorganism.

In a further aspect, the invention provides a method of modifying a microorganism to have resistance to ionic liquids, e.g., ionic liquids where the anion is Cl⁻ or acetate, the method comprising introducing an expression cassette into the microorganism, wherein the expression cassette comprises a gene encoding a SMR polypeptide that has at least 70% identity, or at least 75%, 80%, or 85% identity, to one amino acid sequence of the group consisting of SEQ ID NOs: 3-20. In some embodiments, the SMR polypeptide has at least 90% identity, or at least 95% identity, or at least 99% identity, to one amino acid sequence selected from the group consisting of SEQ ID NOs: 3-20. In other embodiments the polypeptide comprises one amino acid sequence selected from the group consisting of SEQ ID NOs: 3-20. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the method further comprises engineering the microorganism to express a tet repressor that binds to the tet repressor sequence. In some embodiments, the method comprises engineering bacteria, such as Escherichia coli. In other embodiments, the method comprises engineering a yeast or filamentous fungi. In some embodiments, the method comprises integrating the heterologous gene encoding the SMR polypeptide into the genome of the microorganism.

In a further aspect, the invention provides a composition comprising a microorganism of the invention comprising a heterologous SMR gene that is capable of expressing SMR. In some embodiments, the composition is a reaction mixture that comprises an ionic liquid and the microorganism of the invention. In some embodiments, the reaction mixture further comprises biomass.

In an additional aspect, the invention provides a method of increasing the yield of soluble sugar from a biomass, the method comprising incubating biomass pretreated with a microorganism genetically modified to comprise a heterologous SMR gene in an enzymatic hydrolysis reaction. The invention additionally provides a method of increasing the yield from a reaction in which soluble sugars are a source of carbon, e.g., a fermentation reaction that produces a biofuel, such as an alcohol, the method comprising incubating biomass pretreated with ionic liquid with a microorganism of the invention in a reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.

FIG. 1 shows the multiple sequence alignment of the first about fifty-six amino acids of SMR efflux proteins. Multiple sequence alignment was generated using ClustalW. Secondary structure elements indicated on top correspond to those observed in the E. coli EmrE protein (Protein Data Bank identification no. 3b5d). The first two probable transmembrane segments (TMS) are indicated with horizontal lines above the alignments. Asterisks indicate fully conserved residues, colons indicate strongly similar residues, and dots indicate weakly similar residues. Dashes represent gaps. Essential residues (as shown in EmrE [Schuldiner, S. 2007. When biochemistry meets structural biology: the cautionary tale of EmrE. Trends Biochem. Sci. 32:252-258]) are highlighted in black. Conserved residues are highlighted in gray. The identities (ID) and similarities (SM) of AbeS with the corresponding SMR transporter are shown in their respective column. Sequences were obtained from the GenBank database by the use of the following (protein) accession numbers: AbeS, A. baumannii AC0037 (FJ843079) (SEQ ID NO:23); A. baumannii AYE (YP_001713109.1) (SEQ ID NO:23); AB0057 (YP_002319996.1) (SEQ ID NO:23); AB307-0294 (YP_002325052.1) (SEQ ID NO:23); ATCC 17978 (YP_001085323.1) (SEQ ID NO:24); EmrE, E. coli (P23895.1) (SEQ ID NO:25); SsmE, S. marcescens (BAF80121.1) (SEQ ID NO:26); QacE, K. aerogenes (P0AGD0.1) (SEQ ID NO:27); QacEdelta1, Klebsiella pneumoniae (ABF48386.1) (SEQ ID NO:27); QacF, Enterobacter aerogenes (Q9X2N9.1) (SEQ ID NO:28); QacC, Staphylococcus aureus (AAM94143.1) (SEQ ID NO:29); QacH, Staphylococcus saprophyticus (O87868.1) (SEQ ID NO:30); QacG, Staphylococcus sp. strain ST94 (O87866.1) (SEQ ID NO:31); EbrA, Bacillus subtilis (O31792.1) (SEQ ID NO:32); EbrB, B. subtilis (O31791.1) (SEQ ID NO:33); Mmr, Mycobacterium tuberculosis (P95094) (SEQ ID NO:34); YkkC, B. subtilis (Q65KV1.1) (SEQ ID NO:35); Smr-2, Pseudomonas aeruginosa (CAH04647.1) (SEQ ID NO:36); SugE, E. coli (AAQ16658.1) SEQ ID NO:37); and YkkD, B. subtilis (Q65KV0.1) (SEQ ID NO:38).

FIG. 2 shows screening for IL tolerance in E. coli.

FIG. 3 shows identifying bacterial IL tolerance genes.

FIG. 4 shows pump expression rescues E. coli growth in IL.

FIG. 5 shows identifying yeast IL tolerance genes.

FIG. 6 shows the IL-inducible repressor EiLR regulates efflux pump expression in E. coli.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a molecule” includes a plurality of such molecules, and so forth.

The term “about” indicates a value including 10% more than the stated value and 10% less than the stated value. When used to describe a number of nucleotides, it also includes a number of nucleotides one more and/or one fewer than the stated number.

These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.

Definitions

As used herein, the term “Small Multidrug Resistance Family protein” or “SMR protein” are used interchangeably to refer to a membrane protein that functions as a transporter of small molecules, or a regulator thereof. The term encompasses variants and interspecies homologs of the specific polypeptides described herein. A nucleic acid that encodes a SMR protein refers to a gene, cDNA, pre-mRNA, mRNA, and the like, including nucleic acids encoding variants and interspecies homologs of the particular amino acid sequences described herein. Thus, in some embodiments, SMR gene encodes a polypeptide having an amino acid sequence that has at least 40% amino acid sequence identity, or at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 300 or more amino acids, or over the length of the entire polypeptide, to an amino acid sequence selected from the group consisting of SEQ ID NOs:3-20. Any one amino acid sequence selected from the group consisting of SEQ ID NOs: 3-20 provides an illustrative amino acid sequence of a SMR proteins suitable for use in the invention.

As used herein, the term “Major Facilitator Superfamily protein” or “MFS protein” are used interchangeably to refer to a membrane protein that functions as a transporter of small molecules, or a regulator thereof. The term encompasses variants and interspecies homologs of the specific polypeptides described herein. A nucleic acid that encodes a MFS protein refers to a gene, cDNA, pre-mRNA, mRNA, and the like, including nucleic acids encoding variants and interspecies homologs of the particular amino acid sequences described herein. Thus, in some embodiments, MFS gene encodes a polypeptide having an amino acid sequence that has at least 40% amino acid sequence identity, or at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 300 or more amino acids, or over the length of the entire polypeptide, to an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, and 21. Any one amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, and 21 provides an illustrative amino acid sequence of a MFS proteins suitable for use in the invention.

The terms “polynucleotide” and “nucleic acid” are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non-ionic backbones, and non-ribose backbones. Thus, nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase. “Polynucleotide sequence” or “nucleic acid sequence” includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.

The term “substantially identical,” used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 40%, 45%, or 50% sequence identity with a reference sequence. Percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. For example, a polynucleotide encoding a SMR polypeptide may have a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-20.

Two nucleic acid sequences or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.

Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10⁻⁵, and most preferably less than about 10⁻²⁰.

Nucleic acid or protein sequences that are substantially identical to a reference sequence include “conservatively modified variants.” With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are illustrative conservative substitutions for one another. 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) (see, e.g., Creighton, Proteins (1984)).

Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other, or a third nucleic acid, under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60° C. For example, stringent conditions for hybridization, such as RNA-DNA hybridizations in a blotting technique are those which include at least one wash in 0.2×SSC at 55° C. for 20 minutes, or equivalent conditions.

The term “promoter,” as used herein, refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis- and trans-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. Promoters are located 5′ to the transcribed gene, and as used herein, include the sequence 5′ from the translation start codon (i.e., including the 5′ untranslated region of the mRNA, typically comprising 100-200 bp). Most often the core promoter sequences lie within 1-2 kb of the translation start site, more often within 1 kbp and often within 500 bp of the translation start site. By convention, the promoter sequence is usually provided as the sequence on the coding strand of the gene it controls. In the context of this application, a promoter is typically referred to by the name of the gene for which it naturally regulates expression. A promoter used in an expression construct of the invention is referred to by the name of the gene. Reference to a promoter by name includes a wildtype, native promoter as well as variants of the promoter that retain the ability to induce expression. Reference to a promoter by name is not restricted to a particular species, but also encompasses a promoter from a corresponding gene in other species.

A “constitutive promoter” in the context of this invention refers to a promoter that is capable of initiating transcription under most conditions in a cell, e.g., in the absence of an inducing molecule, whereas an “inducible promoter” initiates transcription in the presence of an inducer molecule.

A polynucleotide is “heterologous” to an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a polynucleotide encoding a polypeptide sequence is said to be operably linked to a heterologous promoter, it means that the polynucleotide coding sequence encoding the polypeptide is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety)

“Recombinant nucleic acid” or “recombinant polynucleotide” as used herein refers to a polymer of nucleic acids wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host cell; (b) the sequence may be naturally found in a given host cell, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a recombinant nucleic acid sequence will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid.

The term “operably linked” refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

The term “expression cassette” or “DNA construct” or “expression construct” refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. In the case of expression of transgenes, one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially identical to a sequence of the gene from which it was derived. As explained herein, these substantially identical variants are specifically covered by reference to a specific nucleic acid sequence. One example of an expression cassette is a polynucleotide construct that comprises a polynucleotide sequence encoding a MFS or SMR protein operably linked to a promoter, e.g., its native promoter, where the expression cassette is introduced into a heterologous microorganism. In some embodiments, an expression cassette comprises a polynucleotide sequence encoding a MFS or SMR protein that is targeted to a position in the genome of a microorganism such that expression of the polynucleotide sequence is driven by a promoter that is present in the microorganism.

The terms “optional” or “optionally” as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

As used herein and in the appended claims, the singular “a”, “an” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “host cell” includes a plurality of such host cells.

Introduction

Ionic liquids are used to pretreat biomass to improve the yield of soluble sugars from downstream reactions such as enzymatic hydrolysis. The invention provides methods of genetically modifying microorganisms, e.g., bacteria such as E. coli or yeast, to have resistance to ionic liquids.

A microorganism is typically genetically modified to express a heterologous MFS or SMR protein by introducing an expression cassette that comprises a polynucleotide encoding an MFS or SMR protein operably linked to a promoter into a microbial host cell. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter comprises a repressor binding site for a repressor, e.g., a tet repressor, that dissociates from the binding site when ionic liquids are present in the media in which the host cell is growing.

A microorganism modified as described herein is used in reactions that employ ionic liquids, for example, reactions comprising biomass pretreated with ionic liquids. A genetically modified microorganism as described herein provides for increased yields of soluble sugars from IL-pretreated biomass in comparison to a microorganism that has not been genetically modified to express a heterologous MFS or SMR protein. Further, a microorganism modified to express MFS or SMR can also provide a growth advantage over a contaminating microorganism that may be present in a reaction, such as a fermentation reaction.

Genetically modified microorganisms engineered to express MFS or SMR can also be used for any other reactions in which it is desirable to use a microorganism that is tolerant to ionic liquids. For example, such an organism can be used in pharmaceutical reactions or other reactions where ionic liquids are used as replacement for organic solvents.

MFS and SMR Nucleic Acid Sequences

The invention employs various routine recombinant nucleic acid techniques. Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Many manuals that provide direction for performing recombinant DNA manipulations are available, e.g., Sambrook & Russell, Molecular Cloning, A Laboratory Manual (3rd Ed, 2001); and Current Protocols in Molecular Biology (Ausubel, et al., John Wiley and Sons, New York, 2009, supplements through 2012).

MFS nucleic acid and polypeptide sequences suitable for use in the invention include MFS nucleic acid sequences that encode a MFS polypeptide as illustrated in SEQ ID NO:1, 2, and 21, or a substantially identical variants. Such a variant typically has at least 60%, often at least 70%, or at least 75%, 80%, 85%, or 90% identity to SEQ ID NO:1, 2, and 21.

SMR nucleic acid and polypeptide sequences suitable for use in the invention include SMR nucleic acid sequences that encode a SMR polypeptide as illustrated in SEQ ID NO:3-20, or a substantially identical variants. Such a variant typically has at least 60%, often at least 70%, or at least 75%, 80%, 85%, or 90% identity to SEQ ID NO:3-20.

Various regions of MFS protein are defined. The typical MFS polypeptide has 14 transmembrane domains. One of skill can obtain or identify a MFS variant for use in the invention by using the sequence alignments to identify residues within the conserved sequences that would be expected to retain function as well as residues outside of the conserved regions that would be tolerant to substitution. In some embodiments, the MFS polypeptide comprises one or more, or all, of the 14 transmembrane domains.

Various regions of SMR protein are defined. For example, the typical SMR polypeptide has 4 transmembrane domains. The helical regions of the first two domains are present on amino acids 1-22 and 31-52 for YkkD. An alignment of some of these SMR proteins is shown in FIG. 1. One of skill can obtain or identify a SMR variant for use in the invention by using the sequence alignments to identify residues within the conserved sequences that would be expected to retain function as well as residues outside of the conserved regions that would be tolerant to substitution. In some embodiments, the SMR polypeptide comprises one or more, or all, of the 4 transmembrane domains, and/or one or more of the conserved amino acid residues identified in FIG. 1.

In some embodiments, an MFS or SMR polypeptide may be modified to have a signal sequence. For example, in some embodiments, it may be desirable to employ a signal sequence from the host cell into which the MFS or SMR gene will be introduced.

MFS or SMR activity for conferring resistance to an IL can be assessed using any number of assays. For example, a gene encoding an MFS or SMR protein can be introduced into a microorganism, such as a bacteria, e.g., E. coli, and tested for the ability to grow in the presence of an IL. In the present invention, a microorganism that is resistant to an ionic liquid has improved growth in the presence of the ionic liquid when compared to the same microorganism, i.e., the same genetic background, that has not been modified to express an MFS or SMR polypeptide. In typical embodiments, growth is increased by at least 10%, 20%, 30%, 40%, or 50% or more compared the control, unmodified organism. In some embodiments, the amount of ionic liquid employed in testing is in the range of from about 1% to about to about 20% IL, e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% or 20%, added to the culture medium. In some embodiments, the amount of ionic liquid employed in testing is at least about 0.1% added to the culture medium. In some embodiments, the ionic liquid is [C₂mim]Cl. In the context of this invention, a microorganism modified to express a heterologous MFS or SMR polypeptide is generally resistant to at least 2% ionic liquid, and in some embodiments, is resistant to at least 10% or 20% ionic liquid, e.g., is resistant to at least 20% [C₂mim]Cl. In some embodiments, a microorganism modified to express a heterologous SMR polypeptide is resistant to at least 0.1% ionic liquid.

Isolation or generation of MFS or SMR polynucleotide sequences can be accomplished by any number of techniques well known in the art. In some embodiments, oligonucleotide probes based on the sequences disclosed herein can be used to identify the desired polynucleotide in a cDNA or genomic DNA library from a desired bacterial species. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different bacterial species.

Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using routine amplification techniques. For instance, PCR may be used to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.

Appropriate primers and probes for identifying a MFS or SMR gene from bacterial cells such as Enterobacter cells, can be generated from comparisons of the sequences provided herein. For a general overview of PCR see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990).

MFS or SMR nucleic acid sequences for use in the invention includes genes and gene products identified and characterized by techniques such as hybridization and/or sequence analysis using exemplary nucleic acid sequences, e.g., SEQ ID NOs:1 or 4.

MFS- or SMR-encoding nucleic acid sequences may additionally be codon-optimized for expression in a desired host cell. Methods and databases that can be employed are known in the art. For example, preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. See, e.g., See e.g., Henaut and Danchin in “Escherichia coli and Salmonella,” Neidhardt, et al. Eds., ASM Pres, Washington D.C. (1996), pp. 2047-2066; Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292.

Preparation of Recombinant Vectors

To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of host cells are prepared. Preparation of recombinant vectors is well known in the art. For example, a DNA sequence encoding a MFS or SMR polypeptide can be combined with transcriptional and other regulatory sequences which will direct the transcription of the sequence from the gene in the intended cells. In some embodiments, an expression vector that comprises an expression cassette that comprises the MFS or SMR gene further comprises a promoter operably linked to the MFS or SMR gene. Such a promoter can be an MFS or SMR promoter from the native MFS or SMR gene or a heterologous promoter. In other embodiments, a promoter and/or other regulatory elements that direct transcription of the MFS or SMR gene are endogenous to the microorganism and an expression cassette comprising the MFS or SMR gene is introduced, e.g., by homologous recombination, such that the heterologous MFS or SMR gene is operably linked to an endogenous promoter and is expression driven by the endogenous promoter.

Expression of the MFS or SMR gene can be controlled by a number of regulatory sequences including promoters, which may be either constitutive or inducible; and, optionally, repressor sequences. Regulatory regions include, for example, those regions that contain a promoter and an operator. A promoter is operably linked to the desired nucleic acid sequence, thereby initiating transcription of the nucleic acid sequence via an RNA polymerase enzyme. An operator is a sequence of nucleic acids adjacent to the promoter, which contains a protein-binding domain where a repressor protein can bind. In the absence of a repressor protein, transcription initiates through the promoter. When present, the repressor protein specific to the protein-binding domain of the operator binds to the operator, thereby inhibiting transcription. In this way, control of transcription is accomplished, based upon the particular regulatory regions used and the presence or absence of the corresponding repressor protein. In some embodiments, an SMR gene is operably linked to a nucleotide sequence comprising a tet repressor binding site.

Additional examples of regulatory regions that can be used to regulate expression of a MFS or SMR gene under a desired environmental conditions include lactose promoters (Lac repressor protein changes conformation when contacted with lactose, thereby preventing the Lac repressor protein from binding to the operator) and tryptophan promoters (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator). Another example is the tac promoter. (See deBoer et al. (1983) Proc. Natl. Acad. ScL USA, 80:21-25.) As will be appreciated by those of ordinary skill in the art, these and other expression vectors may be used in the present invention, and the invention is not limited in this respect.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a modified promoter from the gene encoding neutral alpha-amylase in Aspergillus niger In which the untranslated leader has been replaced by an untranslated leader from the gene encoding triose phosphate isomerase in Aspergillus nidulans); and mutant, truncated, and hybrid promoters thereof.

Suitable promoters of use in a yeast host cell include promoters obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

An expression vector may also comprise additional sequences that influence expression of an MFS or SMR gene. Such sequences include enhancer sequences or other sequences such as transcription termination sequences, and the like.

A vector expressing an MFS or SMR gene in accordance with the invention may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

An expression vector of the invention preferably contains one or more selectable markers which permit easy selection of transformed hosts. For example, an expression vector my comprise a gene that confers antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance) to the recombinant host organism, e.g., a bacterial cell such as E. coli, that comprises the vector.

Suitable markers for other microbial host cells, such as yeast host cell are also well known and include, for example, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host include, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Markers for use in Aspergillus include the amdS and pyrG genes of Aspergillus nidulans ox Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus. Markers for use in Trichoderma include bar and amdS.

An expression vector may additionally contain an element(s) that permits integration of the vector into the host's genome. In some embodiments, the expression vector may contain an element that permits autonomous replication of the vector in the cell independent of the genome.

Although any suitable expression vector may be used to incorporate the desired sequences, readily available bacterial expression vectors include, without limitation: plasmids, such as pSC1O1, pBR322, pBBR1MCS-3, pUR, pEX, pMR1OO, pCR4, pBAD24, pUC19; bacteriophages, such as M1 3 phage and λ phage. Of course, such expression vectors may only be suitable for particular host cells. One of ordinary skill in the art, however, can readily determine through routine experimentation whether any particular expression vector is suited for any given host cell. For example, the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector. In addition, reference may be made to the relevant texts and literature, which describe expression vectors and their suitability to any particular host cell.

Expression vectors of the invention may be introduced into the host cell using any number of well-known methods, including calcium phosphate transfection, DEAE-Dextran mediated transfection, electroporation, via lipid complexes, or other common techniques.

Host Cells

Any number of microorganism can be transformed with an expression vector comprising a gene encoding a MFS or SMR protein in accordance with the invention. In some embodiments, the host cell is prokaryotic, such bacterial host cells. Examples of bacterial host cells include, without limitation, species assigned to the Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Clostridium, Enterococcus, Lactobacillus, Lactococcu, Oceanobaciilus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Staphococcus, Strpeotcoccus, Streptomyces, Rhizobia, Vitreoscilla, Synechococcus, Synechocystis, and Paracoccus taxonomical classes. In some embodiments, the prokaryotic host cells are E. coli, Bacillus sp. such as Bacillus subtilis. In some embodiments, the host cells are cyanobacteria.

In some embodiments, the host cell is a yeast. Examples of yeast host cells include Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia host cells. In some embodiments, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis cell. In some embodiments, the yeast host cell is a Kluyveromyces lactis cell. In another embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In other embodiments, the host cell is a filamentous fungal cell. In some embodiments, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Malbranchea, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. For example, a filamentous fungal host cell may be an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In other embodiments, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In further embodiments, the filamentous fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Malbranchea cinnamomea, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma Iongibrachiatum, Trichoderma reesei, or Trichoderma viride cell. In some embodiments, the filamentous fungal host cell is Ustilago maydis.

The host cells of the present invention may be genetically modified in that recombinant nucleic acids have been introduced into the host cells, and as such the genetically modified host cells do not occur in nature. The suitable host cell is one capable of expressing one or more nucleic acid constructs encoding one or more proteins for different functions.

In some embodiments, the host cell may be modified to express a tet repressor. The TetR family of transcription repressors is well known and various TetR sequences are available that can be employed to modify a host cell (see, e.g., Microbiol Mol Biol Rev. 69:326-356, 2005, which is incorporated by reference).

In some embodiments, the host cell naturally produces any of the proteins encoded by the polynucleotides of the invention. The genes encoding the desired proteins may be heterologous to the host cell or these genes may be endogenous to the host cell but are operatively linked to heterologous promoters and/or control regions which result in the higher expression of the gene(s) in the host cell. In other embodiments, the host cell does not naturally produce the desired proteins, and comprises heterologous nucleic acid constructs capable of expressing one or more genes necessary for producing those molecules.

Methods of Using Microorganism Engineered to Express MFS or SMR

The organisms modified in accordance with the invention can be used in saccharfication or fermentation reactions that employ IL-pretreated biomass. Biomass that is pretreated with an IL include, but is not limited to, a cellulose biomass, a hemicellulose biomass, a lignocellulose biomass and mixtures thereof. In some embodiments, the biomass pretreated with an IL is a lignocellulose biomass. The biomass may be pretreated using known processes (see, for example, Hermanutz, et al., Macromol. Symp. 262:23-27, 2008; PCT application PCT/US2012/042790).

Examples of ILs suitable for pretreatment of the biomass and for the hydrolysis of cellulose by cellulases include, but are not limited to 1-ethyl-3-methylimidazolium acetate (EMIM Acetate), l-ethyl-3-methylimidazolium chloride (EMIM CI or ([C₂mim]Cl), 1-ethyl-3-methylimidazolium hydrogensulfate (EMIM HOSO₃), l-ethyl-3-methylimidazolium methylsulfate (EMIM MeOSO₃), 1-ethyl-3-methylimidazolium ethylsulfate (EMIM EtOSO₃), 1-ethyl-3-methylimidazolium methanesulfonate (EMIM MeSO₃), 1-ethyl-3-methylimidazolium tetrachloroaluminate (EMIM AICI4), 1-ethyl-3-methylimidazolium thiocyanate (EMIM SCN), 1-butyl-3-methylimidazolium acetate (BMIM Acetate), l-butyl-3-methylimidazolium chloride (BMIM CI), 1-butyl-3-methylimidazolium hydrogensulfate (BMIM HOSO₃), 1-butyl-3-methylimidazolium methanesulfonate (BMIM MeSO₃), 1-butyl-3-methylimidazolium methylsulfate (BMIM MeOSO₃), 1-butyl-3-methylimidazolium tetrachloroaluminate (BMIM AICI4), l-butyl-3-methylimidazolium thiocyanate (BMIM SCN), 1-ethyl-2,3-dimethylimidazolium ethylsulfate (EDIM EtOSO₃), Tris(2-hydroxyethyl)methylammonium methylsulfate (MTEOA MeOSO₃), 1-methylimidazolium chloride (MIM CI), 1-methylimidazolium hydrogensulfate (MIM HOSO₃), 1,2,4-trimethylpyrazolium methylsulfate, tributylmethylammonium methylsulfate, choline acetate, choline salicylate, and the like. The ionic liquid can comprises one or a mixture of the compounds. In some embodiments, the ionic liquid has an imidazolium cation. Additional suitable ILs are taught in U.S. Pat. No. 6,177,575. It will be appreciated by those of skill in the art that others ILs that will be useful in the process of the present invention are currently being developed or will be developed in the future, and the present invention contemplates their future use.

The pretreated biomass, e.g., the lignocellulose biomass, can be hydrolyzed enzymatically to break down, for example, hemicellulose and/or cellulose, into sugars. Typically, the pretreated biomass is subjected to the action of one, or several or all enzyme activities selected from a cellulase, a cellobiohydrolase, an endoglucanase, a glucano-hydrolase, a protease, a pectinase, a xylanase, a lyase, a ferulic acid esterase, and a mannanase. In one embodiment, the pretreated biomass is subjected to the action of a cellulase, such as a thermostable cellulase. Cellulases suitable for use in the present invention are commercially available from, for example, Genencor (USA) and Novozymes (Europe). For instance, Novozyme has a number of different enzymes and enzyme complexes that are specifically designed to be useful for the hydrolysis of lignocellulosic materials. Examples include, but are not limited to, the following: NS50013, which is a cellulase; NS50010, which is a beta-glucosidase; NS22086, which is a cellulase complex; NS22086, which is a xylanase; NS22118, which is β-glucosidase; NS22119, which is an enzyme complex of carbohydrases, including arabinase, β-glucanase, cellulase, hemicellulase, pectinase, and xylanase; NS22002, which is a mixture of β-glucanase and xylanase; and NS22035, which is a glucoamylase. In addition, suitable thermostable cellulases are disclosed in PCT International Publication No. WO 2010/124266, the teachings of which are incorporated herein by reference. Other hydrolases suitable for hydrolyzing the pretreated biomass, i.e., the lignocellulosic material, will be known to those of skill in the art.

Hydrolysis may additionally be carried out by contacting the pretreated biomass with a microorganism genetically modified to express an MFS or SMR protein in accordance with the invention. Such an organism may be additionally modified to express one or more cellulases, or other enzymes involved in lignocellulose degradation.

A host cell engineered to express MFS or SMR may also be genetically modified to enhance other desired properties, such as improving growth, or modified to enhance yield of a desired product in a reaction that contains ionic liquids.

A host cell modified to express MFS or SMR may be used in any reaction for which it is desired to employ an organism that is tolerant to ionic liquids. For example, such a host cell can be used for producing any fermentation product or other product for which sugars obtained from hydrolysis of an ionic-liquid pretreated biomass can serve as a carbon source. Examples of products include, but are not limited to, alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂); antibiotics (e.g., penicillin and tetracycline); vitamins (e.g., riboflavin, B12, beta-carotene), fatty acids and fatty acid derivatives (as described, e.g., in PCT/US2008/068833); isoprenyl alkanoates (as described, e.g., PCT/US2008/068756, methyl butenol (as described, e.g., PCT/US2008/068831; fatty acid esters (as described, e.g., in PCT/US2010/033299), isoprenoid-based alternative diesel fuels (as described, e.g., in PCT/US2011/059784; a polyketide synthesized by a polyketide synthase, such as a diacid (see, e.g., PCT/US2011/061900), biofuels (see, e.g., PCT/US2009/042132) and alpha-olefins (see, e.g., PCT/US2011/053787). Both the enzymatic hydrolysis and subsequent steps to produce a desired organic compound can be carried out using procedures known to and used by those of skill in the art.

Methods of Identifying Genes that Confer Resistance to Ionic Liquids

A method of screening for genes that confer resistance to ionic liquids is taught in U.S. Patent Application Pub. No. 2014/0038848, which is incorporated by reference. The method comprises preparing a genomic library comprising the genome of a microorganism resistant to ionic liquids, e.g., Enterobacter lignolyticus SCF1 in a vector, e.g., a vector that accommodates large inserts, e.g., at least 30 kb, and introducing the library into the desired host cells. Typically, a low copy number vector is employed to enhance stability of the library. In some embodiments, the vector is a fosmid vector or bacterial artificial chromosome that contains an f-factor origin replication. These are derived from E. coli f-factor which is able to stably integrate large segments of genomic DNA. When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable library. The vector may contain additional sequences such as selectable markers. The library can then be screened for colonies that are resistant to ionic liquids by plating the library onto media containing an ionic liquid of interest, e.g., [C₂mim]Cl, at a desired concentration, e.g., from about 1% to about 6%. Colonies that exhibit relatively fast growth can then be selected and evaluated to determine gene(s) present in the clone and further tested to determine whether the gene can confer resistance to an ionic liquid to host cells transformed with an expression vector that expresses the gene. In some embodiments, the copy number may be amplified to facilitate isolated the DNA of interest.

In some embodiments, the library comprises genomic inserts from more than one microorganism. For example, genomic DNA can be isolated from an environmental sample and a library can be screened as described above. The identity of the microorganisms from which the genomic DNA is isolated need not be known.

YDR090C Proteins

Another aspect of the invention is the S. cerevisiae YDR090C protein. Where this present specification refers to a MFS or SMR protein, that term is meant also include the S. cerevisiae YDR090C protein.

For example, in one aspect, the invention provides a recombinant microorganism having resistance to ionic liquids, e.g., an ionic liquid where the anion is Cl⁻ or acetate, wherein the microorganism comprises a heterologous gene encoding a protein or polypeptide having at least 70% identity, or at least 75%, 80%, or 85% identity, to SEQ ID NO:22. In some embodiments, the polypeptide has at least 90% identity, or at least 95% identity, to SEQ ID NO:22. In some embodiments, the polypeptide comprises SEQ ID NO:22. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the microorganism is also genetically modified to express a tet repressor protein that binds to the tet repressor sequence. In some embodiments, the microorganism is a bacteria, such as Escherichia coli. In other embodiments, the microorganism is a yeast or filamentous fungi. In some embodiments, the heterologous gene encoding the polypeptide is integrated into the genome of the microorganism. In some embodiments, the heterologous gene encoding the polypeptide is present on an extrachromosomal autonomously replicating sequence present in the microorganism.

In a further aspect, the invention provides a method of modifying a microorganism to have resistance to ionic liquids, e.g., ionic liquids where the anion is Cl⁻ or acetate, the method comprising introducing an expression cassette into the microorganism, wherein the expression cassette comprises a gene encoding a protein or polypeptide that has at least 70% identity, or at least 75%, 80%, or 85% identity, to SEQ ID NO:22. In some embodiments, the polypeptide has at least 90% identity, or at least 95% identity, to SEQ ID NO:22. In other embodiments the polypeptide comprises SEQ ID NO:22. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the method further comprises engineering the microorganism to express a tet repressor that binds to the tet repressor sequence. In some embodiments, the method comprises engineering bacteria, such as Escherichia coli. In other embodiments, the method comprises engineering a yeast or filamentous fungi. In some embodiments, the method comprises integrating the heterologous gene encoding the polypeptide into the genome of the microorganism.

In a further aspect, the invention provides a composition comprising a microorganism of the invention comprising a heterologous gene that is capable of expressing a YDR090C polypeptide. In some embodiments, the composition is a reaction mixture that comprises an ionic liquid and the microorganism of the invention. In some embodiments, the reaction mixture further comprises biomass.

In an additional aspect, the invention provides a method of increasing the yield of soluble sugar from a biomass, the method comprising incubating biomass pretreated with a microorganism genetically modified to comprise a heterologous YDR090C gene in an enzymatic hydrolysis reaction. The invention additionally provides a method of increasing the yield from a reaction in which soluble sugars are a source of carbon, e.g., a fermentation reaction that produces a biofuel, such as an alcohol, the method comprising incubating biomass pretreated with ionic liquid with a microorganism of the invention in a reaction.

Salmonella MFS SmvA Pump and SmvR Regulator

The amino acid sequence of the Salmonella MFS Pump, SmvA Pump (found at the website for ncbi.nlm nih gov/protein/AAL20492.1) is:

(SEQ ID NO: 1) 1 mfrgwltlvi ivlvyipvai datvlhvaap tlsmtlgasg nellwiidiy slvmagmvlp 61 mgalgdrigf krllmlggtl fglaslaaaf shtaswliat rvllaigaam ivpatlagir 121 atfceekhrn malgvwaavg sggaafgpli ggillehfyw gsvflinvpi vlvvmgltar 181 yvprgagrrd qplnlghavm liiailllvy saktalkghl slwvisftll tgalllglfi 241 rtqlatsrpm idmrlfthri ilsgvvmamt amitivgfel lmagelqfvh glspyeagvf 301 mlpvmvasgf sgpiagvlvs rlglrlvatg gmalsalsfy glamtdfstq qwqawglmal 361 lgfsaasall astsaimaaa paekaaaaga ietmayelga glgiaifgll lsrsfsasir 421 lpagleagei arasssmgea vqlanslppt qgqaildaar hafiwshsva lssagsmlll 481 lavgmwfsla kaqrr

The amino acid sequence of the Salmonella MFS Pump, SmvR Regulator (found at the website for ncbi.nlm nih gov/protein/AAL20493.1) is:

(SEQ ID NO: 2) 1 msylnrderr evilqaamrv alaegfaamt vrriaseadv aagqvhhhfs sagelkalaf 61 vhlirtllda gqvpppatwr arlhamlgse dggfepyikl wreaqiladr dphirdayll 121 tmqmwheetv tiieqgkqag eftftanatd iawrlialvc gldgmyvlgi pemadpafkf 181 hldrmitlel fa SMR Proteins

The amino acid sequence of the Bacillus licheniformis Pump (from Z98 environmental isolate has the same DNA/protein sequence as the type strain (ATCC 14580=DSM13)), YkkC (found at the website for ncbi.nlm nih gov/protein/AAL20493.1) is:

(SEQ ID NO: 3) 1 mrwgsvilaa lfeigwvmgl khadsalewi ctaaavvmsf yilvkagekl pvgtvyavft 61 glgtagtvvc eialfnepan iaklaligvl lcgviglklv tneekgeas

The amino acid sequence of the Bacillus licheniformis Pump (from Z98 environmental isolate has the same DNA/protein sequence as the type strain (ATCC 14580=DSM13)), YkkD (found at the website for ncbi.nlm.nih gov/protein/AAU40314.1) is:

(SEQ ID NO: 4) 1 mewicliaag ilemlgvtmm nqfhkdkrvr wiflliigfa asffllslam etlpmgtaya 61 vwtgigtvgg alvgilfyge pkdgkriffi alilgsavgl klis

The promoter preceding these Bacillus licheniformis genes may encode an IL-sensing riboswitch region. The environmental Z98 isolate's promoter is similar to, but slightly shorter than, the promoter in the type strain.

The amino acid sequence of the Bacillus cereus JP5 Pump A1 (first member of operon) is:

(SEQ ID NO: 5) MKNRAWLYVILTCIFEIFWVFGFNTADTWWHWIIILGVIAVDFHFLSKA CEHLATGTVYAIFAGAGTVGTFLMDVFLFGGSFSVGKLFFIVMVVAGV IGLKLADNKEESVEGAA*

The amino acid sequence of the Bacillus cereus JP5 Pump A2 (second member of operon) is:

(SEQ ID NO: 6) MGWFFVFCAAISEIVGVIGLKMYSKDKTLANGALYIGGFATSFAFLYTSF LFLQVSVAYAVWIGIGTAGAVLLNMFLFGESKSKARIISVALIVCGVTGL KALS*

The amino acid sequence of the Bacillus cereus JP5 Pump regulator (repressor) is:

(SEQ ID NO: 7) MMNKKEKIVYAAIEVFQEKGVEKTKISDIVKLAGIAQGTFYLYFPSKLSV MPAIAEVMVEKMILAVKEKVQNDAPFSSKVTQVIDAVFHFIAEYREIQAL MYAGLASTEHIKEWEAVYEPLYMWLSEFLNEAKEAGEIRDSVHAERTAKL FIALVESAAEQVYLYDHKDDEQVELQKAEVLDFLTHALHIKK*

The amino acid sequence of the Bacillus cereus JP5 Pump regulator (acetyl transferase) is:

(SEQ ID NO: 8) MNICNAVTSDVKEIYSLIEVYAKEGVVLPRSLLSLYQYLQCLYVVKEEE KIVGVAGLHVLGEDLAEVRSLVVSHIYAGKGIGRMLVNHVINEAAKIKV SRVISLTYETEFFQKCGFDFVNRDALPEKVWIDCRHCPKVDYCDEVAM IRYVG*

The amino acid sequence of the Bacillus thuringiensis BGSC 4A11^(T) Pump A1 (first member of operon) (found at the website for ncbi.nlm.nih.gov/protein/228821582) is:

(SEQ ID NO: 9) 1 mrgmknrawl yviltcifei fwvfgfntad twwhwiiilg viavdfhfls kacehlatgt 61 vyavfagagt vgtflmdvfl fggsfsagkl ffilmvvagv iglkladnke etvegaa

The amino acid sequence of the Bacillus thuringiensis BGSC 4A11^(T) Pump A2 (second member of operon) (found at the website for ncbi.nlm.nih.gov/protein/228821583) is:

(SEQ ID NO: 10) 1 mgwffvfcaa iseivgvigl kmyskdktla ngaiyiggfa tsfaflytsf lflgvsvaya 61 vwigigtaga vllnmflfge skskariisv flivcgvtgl kals

The amino acid sequence of the Bacillus thuringiensis BGSC 4A11^(T) Pump A Regulator (repressor) (found at the website for ncbi.nlm nih gov/protein/228821584) is:

(SEQ ID NO: 11) 1 mmnkkekivy aaievfgekg vektkisdiv klagiaggtf ylyfpsklsv mpaiaevmve 61 kmilavkekv gkdvpfsnkv agvidavfhf ieeyreigal myaglasteh ikeweavyep 121 lymwlsefls eakeageird svhaertakl fialvesaae qvylydh kdd egvelqkaev 181 ldflthalhi kk

The amino acid sequence of the Bacillus thuringiensis BGSC 4A11^(T) Pump A Possible Regulator (acetyl transferase) (found at the website for ncbi.nlm.nih.gov/protein/228821578) is:

(SEQ ID NO: 12) 1 mtsdvkeiyn lievyakegv vlprsllsly qylqclyvmk ederivgvag lhvlgedlae 61 irslvvshty agkgigrmlv nhvmeeatki nvrrvislty etvffqkcgf dfvnketlpe 121 kvwidcrhcp kvdycdevam vryvg

The amino acid sequence of the Bacillus cereus JP5 Pump B1 (first member of operon) is:

(SEQ ID NO: 13) MAWIYVILAGIIEIFWVIGLKHAEAPLEWAGVALLITISFVLLFRAYKDL PVGTVYAVFTGIGAGGIVLTEIFVFGEPFSIVKVLLIGLIFFGVIGLKRV TEEKEAKEAA*

The amino acid sequence of the Bacillus cereus JP5 Pump B2 (second member of operon) is:

(SEQ ID NO: 14) MAWVFLILAGICEIIGVLFMKVATEKKGWAPKVILIANFGVSFFFLSLAM NTLPMGTAYAIWTGIGTAGSALLGILIFRESADWRRLAFLSCILCGAVGL KLLS*

The amino acid sequence of the Bacillus cereus JP5 Pump B regulator (repressor) is:

(SEQ ID NO: 15) MTANRIKAVALSHFARYGYEGTSLANIAQEVGIKKPSIYAHFKGKEELYF TCLESALQKDLQSFTGDIEKFSNSSTEELLLQLLKGYAKRFGESEESMFW LRTSYFPPDAFREQIINKANVHIENVGKLLFPVFKRASEQDELHNIEVKD ALEAFLCLLDGLMVELLYAGLNRFETRLDASWKVFWRGLSN*

The amino acid sequence of the Bacillus thuringiensis BGSC 4A11^(T) Pump B1 (first member of operon) (found at the website for ncbi.nlm.nih.gov/protein/228818299) is:

(SEQ ID NO: 16) 1 mawiyviiag iieifwvigl kkaeaplewa gvallitisf vllfraykdl pvgtvyavft 61 gigaggivlt eififgepfs ivkvlligli ffgviglkrv teekeakeaa

The amino acid sequence of the Bacillus thuringiensis BGSC 4A11^(T) Pump B2 (second member of operon) (found at the website for ncbi.nlm.nih.gov/protein/228818300) is:

(SEQ ID NO: 17) 1 mawvflilag iceiigvlfm kvatekkgwa pkvilianfg vsffflslam ntlpmgtaya 61 iwtgigtags allgilifre sadwrrlafl scilcgavgl klls

The amino acid sequence of the Bacillus thuringiensis BGSC 4A11^(T) Pump B Regulator (repressor) (found at the website for ncbi.nlm nih gov/protein/228818298) is:

(SEQ ID NO: 18) 1 mkmtanrika valshfaryg yegtslania qevgikkpsi yahfkgkeel yficlesalq 61 kdlqsftgdi esfsnsstee lllkllkgya krfgeseesm fwlrtsyfpp dafreqiink 121 anvhienvgk llfpvfkras eqdelhniev kdaleaflcl ldglmvelly aglnrfetrl 181 daswkvfwrg lsn

The amino acid sequence of the E. coli EmrE (found at the website for ncbi.nlm.nih.gov/protein/AAC73644.1) is:

(SEQ ID NO: 19) 1 mnpyiylgga ilaevigttl mkfsegftrl wpsvgtiicy casfwllaqt layiptgiay 61 aiwsgvgivl isllswgffg qrldlpaiig mmlicagvli inllsrstph

The amino acid sequence of the E. coli SugE (found at the website for ncbi.nlm.nih.gov/protein/AAC77108.2) is:

(SEQ ID NO: 20) 1 mswiilviag llevvwavgl kythgfsrlt psvitvtami vsmallawam kslpvgtaya 61 vwtgigavga aitgivllge sanpmrlasl alivlgiigl klsth Yeast MFS Protein

The amino acid sequence of the S. cerevisiae SGE1 (found at the website for yeastgenome.org/cgi-bin/locus.fpl?locus=S000006402) is:

(SEQ ID NO: 21) 1 MKSTLSLTLC VISLLLTLFL AALDIVIVVT LYDTIGIKFH DFGNIGWLVT 51 GYALSNAVFM LLWGRLAEIL GTKECLMISV IVFEIGSLIS ALSNSMATLI 101 SGRVVAGFGG SGIESLAFVV GTSIVRENHR GIMITALAIS YVIAEGVGPF 151 IGGAFNEHLS WRWCFYINLP IGAFAFIILA FCNTSGEPHQ KMWLPSKIKK 201 IMNYDYGELL KASFWKNTFE VLVFKLDMVG IILSSAGFTL LMLGLSFGGN 251 NFPWNSGIII CFFTVGPILL LLFCAYDFHF LSLSGLHYDN KRIKPLLTWN 301 IASNCGIFTS SITGFLSCFA YELQSAYLVQ LYQLVFKKKP TLASIHLWEL 351 SIPAMIATMA IAYLNSKYGI IKPAIVFGVL CGIVGSGLFT LINGELSQSI 401 GYSILPGIAF GSIFQATLLS SQVQITSDDP DFQNKFIEVT AFNSFAKSLG 451 FAFGGNMGAM IFTASLKNQM RSSQLNIPQF TSVETLLAYS TEHYDGPQSS 501 LSKFINTAIH DVFYCALGCY ALSFFFGIFT SSKKTTISAK KQQ*

In some embodiments, the yeast MFS protein has the amino acid sequence of S. cerevisiae SGE1 with the exception of having the following amino acid substitution: S282P, S284L, or both.

Yeast YDR090C Protein

The amino acid sequence of the S. cerevisiae YDR090C (found at the website for yeastgenome.org/cgi-bin/locus.fpl?locus=ydr090C) is:

(SEQ ID NO: 22) 1 MISEKAATAL ATIATVCWCV QLIPQIIYNW KKKDCTGLPP LMMFLWVVSG 51 IPFAIYFCVS KGNVILQVQP HLFMFFCSIS FVQSCYYPPI SMARSKIVMI 101 VAAIIAADVG MEVGFILWLR PLYEKGVKWP DLIFGISASV LLAVGLLPPY 151 FELAKRKGRV IGINFAFLFI DSLGAWLSII SVILGNMDIM GIILYSIVAG 201 MELGIFASHF IWWCRFRFLA KGNTFDEESG QAQKEEPDEK IEQDISKSDR 251 NVTNYNLDNC SIPDDASSFA DDFNIYDSTD GGTLSRAQTL HAVHGVVVRT 301 DPDRYSRLSV *

The present invention provides for the following:

A recombinant microorganism having resistance to ionic liquids, wherein the microorganism comprises a heterologous gene encoding a yeast Major Facilitator Superfamily (MFS), or a Salmonella MFS SmvA Pump or SmvR Regulator, polypeptide operably linked to a promoter, wherein the polypeptide has at least 70% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, and 21. In some embodiments, the polypeptide has at least 90% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, and 21. In some embodiments, the polypeptide comprises one amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, and 21, SEQ ID NO:21 with the following amino acid substitution S282P, S284L, or both. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the microorganism is a bacteria. In some embodiments, the bacteria is Escherichia coli. In some embodiments, the microorganism is a yeast or filamentous fungi. In some embodiments, the anion of the ionic liquid is Cl— or acetate. In some embodiments, the heterologous gene encoding the polypeptide is on a vector or is integrated into the genome of the microorganism.

A method of modifying a microorganism to have resistance to ionic liquids, the method comprising introducing an expression cassette into the microorganism, wherein the expression cassette comprises a gene encoding a yeast MFS, or a Salmonella MFS SmvA Pump or SmvR Regulator, polypeptide that has at least 70% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, and 21. In some embodiments, the polypeptide has at least 90% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, and 21. In some embodiments, the polypeptide comprises one amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, and 21. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the microorganism is a bacteria. In some embodiments, the bacteria is Escherichia coli. In some embodiments, the microorganism is a yeast or filamentous fungi. In some embodiments, the anion of the ionic liquid is Cl— or acetate. In some embodiments, the heterologous gene encoding the polypeptide is on a vector or is integrated into the genome of the microorganism.

A method of increasing the yield of soluble sugar from a biomass, the method comprising incubating biomass pretreated with a recombinant microorganism having resistance to ionic liquids of the present invention in an enzymatic hydrolysis reaction.

A method of increasing the yield from a reaction in which soluble sugars are a source of carbon, the method comprising incubating biomass pretreated with ionic liquid with a recombinant microorganism having resistance to ionic liquids of the present invention. In some embodiments, the biomass is incubated with the microorganism in a fermentation reaction that produces an alcohol.

A composition comprising biomass pretreated with ionic liquid and a recombinant microorganism having resistance to ionic liquids of the present invention.

A recombinant microorganism having resistance to ionic liquids, wherein the microorganism comprises a heterologous gene encoding a Small Multidrug Resistance Family (SMR) polypeptide operably linked to a promoter, wherein the polypeptide has at least 70% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:3-20. In some embodiments, the polypeptide has at least 90% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:3-20. In some embodiments, the polypeptide comprises one amino acid sequence selected from the group consisting of SEQ ID NOs:3-20. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the microorganism is a bacteria. In some embodiments, the bacteria is Escherichia coli. In some embodiments, the microorganism is a yeast or filamentous fungi. In some embodiments, the anion of the ionic liquid is Cl— or acetate. In some embodiments, the heterologous gene encoding the polypeptide is on a vector or is integrated into the genome of the microorganism.

A method of modifying a microorganism to have resistance to ionic liquids, the method comprising introducing an expression cassette into the microorganism, wherein the expression cassette comprises a gene encoding a SMR polypeptide that has at least 70% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:3-20. In some embodiments, the polypeptide has at least 90% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:3-20. In some embodiments, the polypeptide comprises one amino acid sequence selected from the group consisting of SEQ ID NOs:3-20. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the microorganism is a bacteria. In some embodiments, the bacteria is Escherichia coli. In some embodiments, the microorganism is a yeast or filamentous fungi. In some embodiments, the anion of the ionic liquid is Cl— or acetate. In some embodiments, the heterologous gene encoding the polypeptide is on a vector or is integrated into the genome of the microorganism.

A method of increasing the yield of soluble sugar from a biomass, the method comprising incubating biomass pretreated with a recombinant microorganism having resistance to ionic liquids of the present invention in an enzymatic hydrolysis reaction.

A method of increasing the yield from a reaction in which soluble sugars are a source of carbon, the method comprising incubating biomass pretreated with ionic liquid with a recombinant microorganism having resistance to ionic liquids of the present invention. In some embodiments, the biomass is incubated with the microorganism in a fermentation reaction that produces an alcohol.

A composition comprising biomass pretreated with ionic liquid and a recombinant microorganism having resistance to ionic liquids of the present invention.

A recombinant microorganism having resistance to ionic liquids, wherein the microorganism comprises a heterologous gene encoding a Saccharomyces cerevisiae YDR090C polypeptide operably linked to a promoter, wherein the polypeptide has at least 70% identity to SEQ ID NO:22. In some embodiments, the polypeptide has at least 90% identity to SEQ ID NO:22. In some embodiments, the polypeptide comprises SEQ ID NO:22. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the microorganism is a bacteria. In some embodiments, the bacteria is Escherichia coli. In some embodiments, the microorganism is a yeast or filamentous fungi. In some embodiments, the anion of the ionic liquid is Cl— or acetate. In some embodiments, the heterologous gene encoding the polypeptide is on a vector or is integrated into the genome of the microorganism.

A method of modifying a microorganism to have resistance to ionic liquids, the method comprising introducing an expression cassette into the microorganism, wherein the expression cassette comprises a gene encoding a Saccharomyces cerevisiae YDR090C polypeptide that has at least 70% identity to SEQ ID NO:22. In some embodiments, the polypeptide has at least 90% identity to SEQ ID NO:22. In some embodiments, the polypeptide comprises SEQ ID NO:22. In some embodiments, the heterologous gene is operably linked to a tet repressor sequence. In some embodiments, the microorganism is a bacteria. In some embodiments, the bacteria is Escherichia coli. In some embodiments, the microorganism is a yeast or filamentous fungi. In some embodiments, the anion of the ionic liquid is Cl— or acetate. In some embodiments, the heterologous gene encoding the polypeptide is on a vector or is integrated into the genome of the microorganism.

A method of increasing the yield of soluble sugar from a biomass, the method comprising incubating biomass pretreated with a microorganism having resistance to ionic liquids of the present invention in an enzymatic hydrolysis reaction.

A method of increasing the yield from a reaction in which soluble sugars are a source of carbon, the method comprising incubating biomass pretreated with ionic liquid with a microorganism having resistance to ionic liquids of the present invention. In some embodiments, the biomass is incubated with the microorganism in a fermentation reaction that produces an alcohol.

A composition comprising biomass pretreated with ionic liquid and a microorganism having resistance to ionic liquids of the present invention.

The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.

EXAMPLE 1 Ionic Liquid Tolerance in Microbes

Cheap, abundant sugar is an absolute requirement for the commercial viability of microbially produced biofuels. Ionic liquid (IL) treatment is among the best and most environmentally friendly methods for preparing plant biomass for enzymatic breakdown into sugars for microbial growth. However, residual ILs present in these sugars inhibit both Escherichia coli and Saccharomyces cerevisiae growth, thereby preventing efficient biofuel production. To understand and overcome this growth inhibition, we have isolated several environmental microbes—Gram-negative and Gram-positive bacteria and fungi—that can tolerate a common imidazolium-based IL at concentrations well above those that are toxic to our biofuel-producing strains. Using metagenomic screening strategies, we have identified two families of bacterial pumps, and one fungal pump, that provide ionic liquid resistance to E. coli and S. cerevisiae, respectively. These bacterial pumps occur in tandem with transcriptional regulators that appear to adjust pump levels in response to changing IL concentrations. Further studies are aimed at fine tuning these pumps and their regulators for use in biofuel-producing E. coli. The discovery of IL-tolerant communities of microbes has overcome a major hurdle in the use of cellulose-solubilizing solvents, and will advance our progress towards readily and economically converting lignocellulose biomass to biofuels and renewable chemicals.

This study describes six ionic liquid tolerance genes that are shown to operate in bacteria and two genes that are shown to operate in yeast. The bacterial genes consist of an MFS pump and five pumps of the Small Multidrug Resistance (SMR) family, that demonstrated to protect E. coli from [C2mim]-based ILs. The MFS pump is the Salmonella protein SmvA, which, along with its regulator SmvR, is capable of protecting E. coli from harmful levels of [C2mim]Cl and [C2mim][OAc]. The Salmonella homologue of Enterobacter's EilA, SmvA is able to secrete [C2mim]Cl and [C2mim][OAc]. The yeast genes are the Saccharomyces cerevisiae MFS pump, SGE1 (YPR198W), and S. cerevisiae YDR090C.

Bacterial Genes

Three of our new SMR pumps come from members of the genus Bacillus. One is from an environmental B. licheniformis isolate, named Z98, and is produced by the protein products of the genes ykkC and ykkD The second and third pumps come from B. cereus isolate JP5. Each of these pumps is similarly comprised of two SMR proteins. Pumps from related bacilli work with comparable activity. Specifically, the YkkCD pump homologue from type strain of Bacillus licheniformis strain and the homologues of the B. cereus pumps with those from the type strain of Bacillus thuringiensis are tested.

Another two bacterial [C2mim]-resistance pumps are the SMR proteins SugE and EmrE from E. coli. Although not normally induced, when either sugE or emrE is expressed from an inducible promoter, it confers IL resistance.

Different IL-pump regulatory mechanisms are also identified. The B. licheniformis pump's expression is predicted to be regulated by a riboswitch that detects ILs directly. As for the MFS pump EilA, which is regulated by the TetR-family repressor EilR, the B. cereus pumps appear to be regulated by TetR-family repressors. Interestingly, one of these TetR family repressors appears to require the joint action of a neighboring protein, and is predicted to be an acetyl transferase, in order to derepress pump expression.

Yeast Genes

Two yeast genes of interest are identified from two highly ionic liquid tolerant strains of S. cerevisiae, the strains 378604X and YS9. Expression of SGE1 from 378604X leads to high IL tolerance in laboratory S. cerevisiae strains. However, expression of SGE1 from either YS9 or our “laboratory” S. cerevisiae strain, the S288C derivative, BY4741, also increase ionic liquid resistance. Similar results are observed for the other yeast gene of interest, YDR090C. In other words, YDR090C sourced from all 3 strains induced a healthy boost in ionic liquid tolerance, but, for YDR090C, it is believed that YS9 may be the best source of the gene.

Possession of extra copies of these genes leads to increased protection, but it is also believed that specific changes in the amino acid sequences of SGE1 and YDR090C proteins in the ionic liquid tolerance strains, or nucleotide sequences in the promoters of these genes in these strains, may lead to especially increased tolerance in these strains.

Finally, there is indication that these genes are be more effective when other nearby genomic regions are included with them. These include, for SGE1, the ˜17 gene region between YPR182W and YPR201W and, for YDR090C, the region encompassing the genes STN1, RRP8, TVP23, AFRI, SSS1, RRP1, SLUT, and YDR089W, as well as the genomic stretches that punctuate these genes.

FIG. 2 shows screening for IL tolerance in E. coli. FIG. 3 shows identifying bacterial IL tolerance genes. FIG. 4 shows pump expression rescues E. coli growth in IL. E. coli cells carrying MFS or SMR pumps driven by an IPTG-inducible promoter were grown in 125 mM (1.8%) [C2mim]Cl and 100 μM IPTG. Many identified pump genes appear beside genes for TetR-type regulatory proteins. FIG. 5 shows identifying yeast IL tolerance genes. S. cerevisiae cells, either wildtype or carrying low-copy number fosmids, were grown in 125 mM (2.1%) [C2mim][OAc] in SC-URA at pH 5. FIG. 6 shows the IL-inducible repressor EiLR regulates efflux pump expression in E. coli. Panel A shows EilR repressor is IL responsive: E. coli cells expressing MFS pump-GFP fusion from native gene cassette. Upper: no [C2mim]Cl. Lower: 4% [C2mim]Cl. Panel B shows EilR enables dynamically pump expression: Single cell fluorescence of E. coli expressing MFS pump-GFP fusion. Panel C shows EilR increases cell growth: Maximum growth rates of E. coli expressing MFS pump constitutively from native eilA promoter or under the control of EilR.

These findings are novel or unexpected for a number of reasons:

(1) This is the first report of specific genes that lead to ionic liquid tolerance in yeast.

(2) These results show that an entirely different class of transporters, SMR pumps are capable of the same activity. Similarly, the yeast gene YDR090C, is of unknown function, but has been implicated in possibly protecting the integrity of the yeast vacuole. This, if true, would also be a novel protective IL protection mechanism. (3) It is surprising that pumps from bacilli, which are Gram-positive, work so well in the membranes of E. coli, which are Gram-negative organisms, as these two classes of bacteria have very different membranes. (4) Unlike the other IL pumps, SugE and EmrE are endogenous to E. coli, and SGE1 and YDR090C are endogeonous to S. cerevisiae. Accordingly, they may prove to be most effective at protecting their native hosts without interfering with the cells' other activities. (5) MFS and SMR pumps may not be very selective about the compounds they export. The regulators that govern their expression, however, are selective about what they respond to. In addition to several new pumps, we have also identified new IL-pump regulatory mechanisms. These identifications may prove even more useful than the pump library for engineering bacterial growth on IL-treated biomass. While pumps may work equally well with different ILs, regulators may be more specialized.

CONCLUSION

Targeted, functional screening of DNA from environmental microorganisms is a powerful approach for the discovery of mechanisms underpinning phenotypes such as bacterial resistance to inhibitory compounds. Such mechanisms, which include substrate specific regulators, remain fully functional when transferred to industrial host organisms, such as E. coli and can be a powerful tool for strain engineering. The discovery and investigation of IL-tolerant communities of microbes has overcome a major hurdle in the use of cellulose-solubilizing solvents, and advances our progress towards readily and economically converting lignocellulose biomass to biofuels and renewable chemicals.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. 

What we claim is:
 1. A culture medium comprising an ionic liquid and a recombinant microorganism, wherein the microorganism comprises a heterologous gene encoding a polypeptide operably linked to a promoter, wherein the polypeptide has at least 95% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:1 and 21, and the expression of the polypeptide confers to the microorganism resistance to the concentration of the ionic liquid in the culture medium.
 2. The culture medium of claim 1, wherein the polypeptide has at least 99% identity to one amino acid sequence selected from the group consisting of SEQ ID NOs:1 and
 21. 3. The culture medium of claim 1, wherein the polypeptide comprises at least 95% identity to SEQ ID NO:21 and the polypeptide comprises a proline at a position corresponding to position 282 of SEQ ID NO:21, a leucine at a position corresponding to position 284 of SEQ ID NO:21, or both.
 4. The culture medium of claim 1, wherein the heterologous gene is operably linked to a tet repressor sequence.
 5. The culture medium of claim 1, wherein the microorganism is a bacteria.
 6. The culture medium of claim 5, wherein the bacteria is Escherichia coli.
 7. The culture medium of claim 1, wherein the microorganism is a yeast or filamentous fungi.
 8. The culture medium of claim 1, wherein the anion of the ionic liquid is Cl⁻ or acetate.
 9. The culture medium of claim 1, wherein the heterologous gene encoding the polypeptide is on a vector or is integrated into the genome of the microorganism.
 10. The culture medium of claim 1, wherein the culture medium further comprises a biomass.
 11. The culture medium of claim 1, wherein the culture medium comprises about 1% to about 20% of the ionic liquid. 